Human monoclonal antibodies specific for cd22

ABSTRACT

Disclosed herein are isolated human monoclonal antibodies that specifically bind human CD22 with a dissociation constant (K d ) of 25 nM or less. Nucleic acids encoding these antibodies, expression vectors including these nucleic acid molecules, and isolated host cells that express the nucleic acid molecules are also disclosed. The antibodies can be used to detect human CD22 in a sample. In some cases, CD22 is soluble CD22. Methods of diagnosing a B-cell malignancy, or confirming a B-cell malignancy diagnosis, are disclosed herein that utilize these antibodies. Methods of treating a subject with a B-cell malignancy are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 15/012,023,filed Feb. 1, 2016, which is a divisional of U.S. patent applicationSer. No. 13/959,061, filed Aug. 5, 2013, issued as U.S. Pat. No.9,279,019 on Mar. 8, 2016, which is a divisional of U.S. applicationSer. No. 12/934,214, filed Sep. 23, 2010, issued as U.S. Pat. No.8,591,889, on Nov. 26, 2013 which is the U.S. National Stage ofInternational Application No. PCT/US2009/039080, filed Apr. 1, 2009,published in English under PCT Article 21(2), which claims the benefitof U.S. Provisional Application No. 61/042,329, filed Apr. 4, 2008. Allof the above-listed applications are herein incorporated by reference intheir entirety.

FIELD

This disclosure relates to monoclonal antibodies, particularly humanmonoclonal antibodies that specifically bind CD22, and their use.

BACKGROUND

CD22 is a 135 kDa sialoglycoprotein expressed on the surface of B-cellsat the mature stages of cell differentiation (Dorken et al., J. Immunol.136:4470-4479, 1986). CD22 functions in B-cell activation and as anadhesion molecule, mediating interactions with activated blood cells andaccessory cells (Hanasaki et al., J. Biol. Chem. 270(13):7533-7542,1995). CD22 is not found on the surface of B cells during the earlystages of development, nor is it expressed in stem cells. However,60-70% of all B-cell lymphomas and leukemias express CD22.

Expression of CD22 in a number of B-cell malignancies has made CD22 anattractive target for immunotherapy. For example, Epratuzumab, ahumanized anti-CD22 antibody has been evaluated in patients withaggressive non-Hodgkin's lymphoma (Leonard et al., Clin. Cancer Res.10:5327-5334, 2004). Immunoconjugates including chimeric (humanized)anti-CD22 antibodies have also been studied, such as CMC-544 (DiJosephet al., Clin. Cancer Res. 12(1):242-249, 2006). An anti-CD22 immunotoxinBL22, a mouse anti-CD22 antibody linked to Pseudomonas exotoxin PE38,has been evaluated for efficacy in the treatment of hairy cell leukemia(Kreitman et al., N. Engl. J. Med. 345(4):241-247; U.S. PatentApplication Publication Nos. 2005/0118182 and 2007/0189962).

CD22-specific antibodies have potential as cancer therapeutic anddiagnostic reagents. However, a major limitation in the clinical use ofmouse monoclonal antibodies is the development of a human anti-murineantibody (HAMA) response. The HAMA response can involve allergicreactions and an increased rate of clearance of the administeredantibody from the serum. A need remains for fully human antibodies thatspecifically bind CD22 with high affinity, which can be used in thediagnosis and treatment of cancer, particularly in the treatment ofB-cell malignancies. However, it is difficult to produce fully humanantibodies to human antigens, as these antigens are generally recognizedas self antigens.

SUMMARY

Isolated human monoclonal antibodies specific for human CD22 areprovided herein. The human monoclonal antibodies bind CD22 with adissociation constant (K_(d)) of about 25 nM or less. In someembodiments, the K_(d) is about 2 to about 20 nM. In some examples, theK_(d) is about 20 nM. In other examples, the K_(d) is about 2 nM.Further provided are compositions including the CD22-specificantibodies, nucleic acids encoding these antibodies, expression vectorscomprising the nucleic acids, and isolated host cells that express thenucleic acids.

Also provided are immunoconjugates comprising the human monoclonalantibodies specific for human CD22. Compositions comprising theimmunoconjugates are also provided.

The antibodies and compositions provided herein can be used for avariety of purposes, such as for diagnosing and for confirming thediagnosis of a B-cell malignancy in a subject. Thus, provided herein isa method of confirming the diagnosis of a B-cell malignancy in asubject, comprising contacting a sample from the subject diagnosed witha B-cell malignancy with a human monoclonal antibody that specificallybinds CD22, and detecting binding of the antibody to the sample. Anincrease in binding of the antibody to the sample relative to binding ofthe antibody to a control sample confirms the B-cell malignancydiagnosis. In some examples, the antibody binds soluble CD22 in thesample. In some embodiments, the methods further comprise contacting asecond antibody that specifically recognizes the CD22-specific antibodywith the sample, and detecting binding of the second antibody.

Similarly, provided herein is a method of detecting a B-cell malignancyin a subject, comprising contacting a sample from the subject with ahuman monoclonal antibody that specifically binds CD22, and detectingbinding of the antibody to the sample. An increase in binding of theantibody to the sample relative to a control sample detects a B-cellmalignancy in the subject. In some examples, the antibody binds solubleCD22 in the sample. In some embodiments, the methods further comprisecontacting a second antibody that specifically recognizes theCD22-specific antibody with the sample, and detecting binding of thesecond antibody.

Further provided is a method of treating a subject diagnosed with aB-cell malignancy, comprising administering a therapeutically effectiveamount of a human CD22-specific monoclonal antibody, or animmunoconjugate comprising the antibody, to the subject.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a series of digital images of electrophoretic gels of m971and m972 in Fab, scFv and IgG1 formats. Antibodies in the indicatedformats were run on 4-12% gradient SDS-PAGE gels under reducingconditions and stained with Coomassie blue. The heavy and light chainsof m971 have a different molecular weight, whereas the heavy and lightchains of m972 ran at the same position. M=molecular weight marker.

FIGS. 1B-1D are line graphs showing binding of m971 and m972 to solubleCD22. Fully human CD22 antibodies in Fab (FIG. 1B), IgG1 (FIG. 1C) andscFv (FIG. 1D) formats were purified and tested for binding to solubleCD22 by ELISA. In FIG. 1D, m972 scFv was tested in comparison to theestablished CD22-specific HA22 scFv.

FIG. 1E is a bar graph of a competition ELISA comparing m971, m972 andHA22. ScFv HA22, Fab m972 and Fab m971 (50 ng/well) were coated to theELISA plate and a mixture of CD22-Fc (1 μg/well) and one of theindicated antibodies was added to each well. The amount of CD22-Fc boundby the coated scFv or Fab was then detected using goat anti-humanFc-IgG-HRP. PC=no competitors added.

FIG. 2 is a schematic diagram illustrating epitope determination ofhuman anti-CD22 antibodies. Sub-domains of CD22 were expressedtransiently and the supernatant from transfected cells was added toELISA plate wells coated with the three indicated antibodies. The boundCD22 domains were detected with mouse anti-Myc-IgG-HRP. The seven boxesrepresent the seven Immunoglobulin-like domains of CD22. Numbers in thebrackets indicate the starting and ending amino acid residues of eachdomain construct. The symbol “+” indicates binding that is approximately10% of binding represented by the symbol “+++”. The symbol “−” indicatesno binding. TM=trans-membrane sequence.

FIGS. 3A-3H are FACS plots showing detection of human CD22 antibodybinding to B lymphocytes. m971, m972 and a weak CD22 binder in IgG1format were used in flow cytometry to test their ability to recognizenative, cell surface-associated CD22. HA22 in scFv format was used asthe positive control. M396, a severe acute respiratory syndrome (SARS)virus-spike specific human IgG1 was used as the isotype control. Anunrelated scFv was used as a negative control (NC) for HA22. SupT1 cells(FIG. 3A and FIG. 3B), which are T lymphocytes that do not express CD22,were used as the negative control cell line. Raji (FIG. 3C and FIG. 3D),BJAB (FIG. 3E and FIG. 3F) and ST486 (FIG. 3G and FIG. 3H) cells, whichare B lymphoma cell lines, were used as the testing cell lines. In eachhistogram, cell count is shown on the y-axis and fluorescence intensityis shown on the x-axis. For each cell line, the upper panel (FIGS. 3A,3C, 3E and 3G) shows the binding by scFv HA22 and the lower panel (FIGS.3B, 3D, 3F and 3H) shows the binding by each human IgG1 antibody. Forexperiments with SupT1 and Raji cells, 10 μg/ml of each of HA22, m971,m972 and the weak CD22 binder were used. For experiments with BJAB andST486 cells, the HA22 concentration remained the same, while 100 and 50μg/ml were used for m971 and m972, respectively.

FIGS. 4A and 4B are a set of FACS plots and a line graph showingaffinity determination of m971 and m972. (FIG. 4A) Increasingconcentrations of both IgG1 m971 and IgG1 m972 were incubated with BJABcells in a flow cytometry assay as described in Example 1 below. (FIG.4B) The median fluorescence intensities from flow cytometry were plottedagainst the corresponding concentrations used for IgG1 m971 and IgG1m972.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. The Sequence Listing is submitted as an ASCII textfile, created on Feb. 1, 2017, 32.7 KB, which is incorporated byreference herein. In the accompanying sequence listing:

SEQ ID NO: 1 is the amino acid sequence of the heavy chain ofCD22-specific antibody m972.

SEQ ID NO: 2 is the amino acid sequence of the light chain ofCD22-specific antibody m972.

SEQ ID NO: 3 is the amino acid sequence of the heavy chain ofCD22-specific antibody m971.

SEQ ID NO: 4 is the amino acid sequence of the light chain ofCD22-specific antibody m971.

SEQ ID NOs: 5 and 6 are the nucleotide and amino acid sequences,respectively, of human CD22, deposited under Genbank™ Accession No.NM_001771 on Sep. 13, 2007.

SEQ ID NO: 7 is the nucleotide sequence encoding the heavy chain ofCD22-specific antibody m972.

SEQ ID NO: 8 is the nucleotide sequence encoding the light chain ofCD22-specific antibody m972.

SEQ ID NO: 9 is the nucleotide sequence encoding the heavy chain ofCD22-specific antibody m971.

SEQ ID NO: 10 is the nucleotide sequence encoding the light chain ofCD22-specific antibody m971.

SEQ ID NO: 11 is the amino acid sequence of a peptide linker.

DETAILED DESCRIPTION I. Abbreviations

3SR: Self-sustained sequence replication

ADCC: Antibody-dependent cell-mediated cytotoxicity

CDR: Complementarity determining region

CLL: Chronic lymphocytic leukemia

DT: Diphtheria toxin

ELISA: Enzyme-linked immunosorbent assay

EM: Effector molecule

FACS: Fluorescence-activated cell sorting

FBS: Fetal bovine serum

FITC: Fluorescein isothiocyanate

HCL: Hairy cell leukemia

HRP: Horseradish peroxidase

Ig: Immunoglobulin

LCR: Ligase chain reaction

LDH: Lactate dehydrogenase

NHL: Non-Hodgkin's lymphoma

mAb: Monoclonal antibody

MALT: Mucosa-associated lymphoid tissue

MPBS: Milk/PBS

PAGE: Polyacrylamide gel electrophoresis

PBMC: Peripheral blood mononuclear cells

PBS: Phosphate-buffered saline

PBST: PBS-Tween 20

PCR: Polymerase chain reaction

PE: Pseudomonas exotoxin

RIA: Radioimmunoassay

sCD22: Soluble CD22

SDS: Sodium dodecyl sulfate

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage.

Definitions of common terms in molecular biology may be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen, such as CD22 or a fragment thereof.Antibodies are composed of a heavy and a light chain, each of which hasa variable region, termed the variable heavy (V_(H)) region and thevariable light (V_(L)) region. Together, the V_(H) region and the V_(L)region are responsible for binding the antigen recognized by theantibody.

Antibodies include intact immunoglobulins and the variants and portionsof antibodies well known in the art, such as Fab fragments, Fab′fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), anddisulfide stabilized Fv proteins (“dsFv”). A scFv protein is a fusionprotein in which a light chain variable region of an immunoglobulin anda heavy chain variable region of an immunoglobulin are bound by alinker, while in dsFvs, the chains have been mutated to introduce adisulfide bond to stabilize the association of the chains. The term alsoincludes genetically engineered forms such as chimeric antibodies (forexample, humanized murine antibodies), heteroconjugate antibodies (suchas, bispecific antibodies). See also, Pierce Catalog and Handbook,1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology,3^(rd) Ed., W. H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs.” The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The Kabat database is now maintainedonline. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species, such as humans.The framework region of an antibody, that is the combined frameworkregions of the constituent light and heavy chains, serves to positionand align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds CD22 will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (i.e. differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds CD22.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, i.e., at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, except theCDRs, are substantially identical to corresponding parts of naturalhuman immunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (see for example, U.S. Pat.No. 5,585,089).

A “human” antibody (also called a “fully human” antibody) is an antibodythat includes human framework regions and all of the CDRs from a humanimmunoglobulin. In one example, the framework and the CDRs are from thesame originating human heavy and/or light chain amino acid sequence.However, frameworks from one human antibody can be engineered to includeCDRs from a different human antibody. All parts of a humanimmunoglobulin are substantially identical to corresponding parts ofnatural human immunoglobulin sequences.

B-cell malignancy: As used herein “B-cell malignancy” includes any typeof leukemia or lymphoma of B cells. B-cell malignancies include, but arenot limited to, non-Hodgkin's lymphoma, Burkitt's lymphoma, smalllymphocytic lymphoma, primary effusion lymphoma, diffuse large B-celllymphoma, splenic marginal zone lymphoma, MALT (mucosa-associatedlymphoid tissue) lymphoma, hairy cell leukemia, chronic lymphocyticleukemia and B-cell prolymphocytic leukemia.

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al. (Mol. Immunol., 16:101-106, 1979). Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In another embodiment, a high binding affinity ismeasured by a competition radioimmunoassay. In another embodiment,binding affinity's measured by ELISA. In one embodiment, the antibodiesbind CD22 with a dissociation constant (K_(d)) of about 25 nM or less.In several embodiments, the human monoclonal antibodies bind human CD22with a binding affinity of about 20 nM or less, about 19 nM or less,about 18 nM or less, about 17 nM or less, about 16 nM or less, or about15 nM or less. In other embodiments, the human monoclonal antibodiesbind human CD22 with a binding affinity of about 10 nM or less, about 9nM or less, about 8 nM or less, about 7 nM or less, about 6 nM or less,or about 5 nM or less. In other embodiments, the human monoclonalantibodies bind human CD22 with a binding affinity of about 4 nM orless, about 3 nM or less, about 2.5 nM or less, about 2 nM or less,about 1.5 nM or less, or about 1 nM or less. In other embodiments, thehuman monoclonal antibodies bind human CD22 with a binding affinity ofabout 1 nM or less, about 0.75 nM or less, about 0.5 nM or less, about0.25 nM or less, or about 0.1 nM or less. In one example, the bindingaffinity is about 20 nM. In another example, the binding affinity isabout 2 nM. As used herein, a binding affinity of “about 20 nM” includesbinding affinities of 19 to 21 nM. Similarly, a binding affinity of“about 2 nM” includes binding affinities of 1 nM to 3 nM.

Biological sample: A sample obtained from cells, tissue or bodily fluidof a subject, such as peripheral blood, cerebrospinal fluid, serum, bonemarrow, urine, saliva, tissue biopsy, surgical specimen, amniocentesissamples and autopsy material. A biological sample is also referred toherein as a “sample.”

Body fluid: Includes any bodily fluid from an animal, for instanceblood, serum, semen, urine, cerebrospinal fluid or saliva.

CD22: A lineage-restricted B cell antigen belonging to theimmunoglobulin superfamily. CD22 is expressed in 60-70% of B celllymphomas and leukemias. CD22 is not present on the cell surface in theearly stages of B cell development or on stem cells. As used herein“CD22” refers to a CD22 polypeptide or variant or fragment thereof.Sequences of human CD22 are known in the art (see, for example Torres etal., J. Immunol. 149(8):2641-2649, 1992; and Wilson et al., J. Exp. Med.173(1):137-146, 1991). Exemplary CD22 nucleotide and amino acidsequences also are provided herein as SEQ ID NO: 5 and SEQ ID NO: 6,respectively. The term “CD22” also includes soluble forms of CD22,referred to as sCD22.

Chemotherapeutic agents: Any chemical agent with therapeutic usefulnessin the treatment of diseases characterized by abnormal cell growth. Suchdiseases include tumors, neoplasms, and cancer as well as diseasescharacterized by hyperplastic growth such as psoriasis. In oneembodiment, a chemotherapeutic agent is an agent of use in treating aB-cell malignancy, such as non-Hodgkin's lymphoma, hairy cell leukemiaor chronic lymphocytic leukemia. In one embodiment, a chemotherapeuticagent is a radioactive compound. One of skill in the art can readilyidentify a chemotherapeutic agent of use (see for example, Slapak andKufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principlesof Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17in Abeloff, Clinical Oncology 2^(nd) ed., © 2000 Churchill Livingstone,Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide toChemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D. S.,Knobf, M. F., Durivage, H. J. (eds): The Cancer Chemotherapy Handbook,4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy isthe administration of more than one agent to treat cancer. One exampleis the administration of an antibody that binds CD22 or a fragmentthereof used in combination with a radioactive or chemical compound.

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disordercharacterized by lymphocytosis, lymphadenopathy, and organomegaly. Itcan be conceived of as a lymphoma that involves the peripheral blood.

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially affect or decrease the affinityof an antibody to CD22. For example, a human antibody that specificallybinds CD22 can include at most about 1, at most about 2, at most about5, and most about 10, or at most about 15 conservative substitutions andspecifically bind the original CD22 polypeptide. The term conservativevariation also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid, provided that antibody specificallybinds CD22. Non-conservative substitutions are those that reduce anactivity or binding to CD22.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Complementarity determining region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively.

Contacting: Placement in direct physical association; includes both insolid and liquid form.

Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to thecells intended to be targeted, as opposed to the cells of the rest of anorganism. In one embodiment, in contrast, the term “toxicity” refers totoxicity of an immunotoxin to cells other than those that are the cellsintended to be targeted by the targeting moiety of the immunotoxin, andthe term “animal toxicity” refers to toxicity of the immunotoxin to ananimal by toxicity of the immunotoxin to cells other than those intendedto be targeted by the immunotoxin.

Degenerate variant (of CD22): A polynucleotide encoding a CD22polypeptide or an antibody that binds CD22 that includes a sequence thatis degenerate as a result of the genetic code. There are 20 naturalamino acids, most of which are specified by more than one codon.Therefore, all degenerate nucleotide sequences are included as long asthe amino acid sequence of the CD22 polypeptide or antibody that bindsCD22 encoded by the nucleotide sequence is unchanged.

Detecting (or detection): Refers to quantitatively or qualitativelydetermining the presence of a biomolecule under investigation. Forexample, in one embodiment, detecting a B-cell malignancy includesquantitatively or qualitatively determining the presence of soluble CD22in a sample.

Diagnosing: Diagnosing or diagnosis of a B-cell malignancy includes bothdetecting a B-cell malignancy and identifying a B-cell malignancy, forexample identifying a B-cell malignancy as non-Hodgkin's lymphoma, hairycell leukemia or chronic lymphocytic leukemia.

Effector molecule (EM): The portion of a chimeric molecule that isintended to have a desired effect on a cell to which the chimericmolecule is targeted. Effector molecule is also known as an effectormoiety, therapeutic agent, or diagnostic agent, or similar terms. Insome embodiments disclosed herein, the EM is a toxin or a detectablelabel.

Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides. Alternatively, the molecule linkedto a targeting moiety, such as an anti-CD22 antibody, may be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (such as anantisense nucleic acid), or another therapeutic moiety that can beshielded from direct exposure to the circulatory system. Means ofpreparing liposomes attached to antibodies are well known to those ofskill in the art (see, for example, U.S. Pat. No. 4,957,735; and Connoret al., Pharm. Ther. 28:341-365, 1985). Diagnostic agents or moietiesinclude radioisotopes and other detectable labels. Detectable labelsuseful for such purposes are also well known in the art, and includeradioactive isotopes such as ³²P ¹²⁵I, and ¹³¹I, fluorophores,chemiluminescent agents, and enzymes.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide, such as CD22.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Framework region: Amino acid sequences interposed between CDRs. Includesvariable light and variable heavy framework regions. The frameworkregions serve to hold the CDRs in an appropriate orientation for antigenbinding.

Hairy cell leukemia (HCL) is a malignant disorder of small B-lymphocytesthat gets its name from the presence of cytoplasmic projections in thesecells. Subjects with HCL commonly present with pancytopenia,splenomegaly and marrow fibrosis. The peripheral blood usually containsa small number of hairy cells. Hairy cells proliferate in the red pulpof the spleen, so splenomegaly is common.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Human anti-murine antibody (HAMA) response: An immune response in ahuman subject to the variable and constant regions of a murine antibodythat has been administered to the patient. Repeated antibodyadministration may lead to an increased rate of clearance of theantibody from the patient's serum and may also elicit allergic reactionsin the patient.

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”), such as CD22. In one embodiment, an immune response is a Tcell response, such as a CD4+ response or a CD8+ response. In anotherembodiment, the response is a B cell response, and results in theproduction of specific antibodies.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody. The effector molecule can be a detectable label or a toxin.Specific, non-limiting examples of toxins include, but are not limitedto, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38,and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxinsthereof, or other toxic agents that directly or indirectly inhibit cellgrowth or kill cells. For example, PE and DT are highly toxic compoundsthat typically bring about death through liver toxicity. PE and DT,however, can be modified into a form for use as an immunotoxin byremoving the native targeting component of the toxin (such as the domainIa of PE and the B chain of DT) and replacing it with a differenttargeting moiety, such as an antibody. A “chimeric molecule” is atargeting moiety, such as a ligand or an antibody, conjugated (coupled)to an effector molecule. The term “conjugated” or “linked” refers tomaking two polypeptides into one contiguous polypeptide molecule. In oneembodiment, an antibody is joined to an effector molecule. In anotherembodiment, an antibody joined to an effector molecule is further joinedto a lipid or other molecule to a protein or peptide to increase itshalf-life in the body. The linkage can be either by chemical orrecombinant means. In one embodiment, the linkage is chemical, wherein areaction between the antibody moiety and the effector molecule hasproduced a covalent bond formed between the two molecules to form onemolecule. A peptide linker (short peptide sequence) can optionally beincluded between the antibody and the effector molecule. Becauseimmunoconjugates were originally prepared from two molecules withseparate functionalities, such as an antibody and an effector molecule,they are also sometimes referred to as “chimeric molecules.” The term“chimeric molecule,” as used herein, therefore refers to a targetingmoiety, such as a ligand or an antibody, conjugated (coupled) to aneffector molecule.

Immunogenic peptide: A peptide, such as a CD22 peptide, which comprisesan allele-specific motif or other sequence, such as an N-terminalrepeat, such that the peptide will bind an MHC molecule and induce acytotoxic T lymphocyte (“CTL”) response, or a B cell response (e.g.antibody production) against the antigen from which the immunogenicpeptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide. In one specific non-limitingexample, an immunogenic polypeptide includes a region of CD22, or afragment thereof, wherein the polypeptide that is expressed on the cellsurface of a host cell that expresses the full-length CD22 polypeptide.

Immunogenic composition: As used herein, an immunogenic composition is acomposition comprising a CD22 polypeptide that induces a measurable CTLresponse against cells expressing CD22 polypeptide, or induces ameasurable B cell response (such as production of antibodies) against aCD22 polypeptide. It further refers to isolated nucleic acids encoding aCD22 polypeptide that can be used to express the CD22 polypeptide (andthus be used to elicit an immune response against this polypeptide). Forin vitro use, an immunogenic composition may consist of the isolatedprotein or peptide epitope. For in vivo use, the immunogenic compositionwill typically comprise the protein or immunogenic peptide inpharmaceutically acceptable carriers, and/or other agents. Anyparticular peptide, such as a CD22 polypeptide, or nucleic acid encodingthe polypeptide, can be readily tested for its ability to induce a CTLor B cell response by art-recognized assays. Immunogenic compositionscan include adjuvants, which are well known to one of skill in the art.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope to a detectably greater degree than, and/or to thesubstantial exclusion of, binding to substantially all other epitopes.Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane (Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, 1988) for a description of immunoassay formatsand conditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, pH) that are typical insidea living mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (i.e., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Isolated: An “isolated” biological component, such as a nucleic acid,protein (including antibodies) or organelle, has been substantiallyseparated or purified away from other biological components in theenvironment (such as a cell) in which the component naturally occurs,i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins andorganelles. Nucleic acids and proteins that have been “isolated” includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes. In one example, a “labeled antibody” refers to incorporationof another molecule in the antibody. For example, the label is adetectable marker, such as the incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (for example, streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionucleotides (such as ³⁵S or ¹³¹I), fluorescentlabels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanidephosphors), enzymatic labels (such as horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (such as a leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags), or magnetic agents, such as gadolinium chelates. In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

Linker: In some cases, a linker is a peptide within an antibody bindingfragment (such as an Fv fragment) which serves to indirectly bond thevariable heavy chain to the variable light chain. “Linker” can alsorefer to a peptide serving to link a targeting moiety, such as anantibody, to an effector molecule, such as a cytotoxin or a detectablelabel.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an scFv. In the specific context, the terms include reference tojoining a ligand, such as an antibody moiety, to an effector molecule.The linkage can be either by chemical or recombinant means. “Chemicalmeans” refers to a reaction between the antibody moiety and the effectormolecule such that there is a covalent bond formed between the twomolecules to form one molecule.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Major histocompatibility complex (MHC): Generic designation meant toencompass the histocompatibility antigen systems (class I and class II)described in different species, including the human leukocyte antigens(“HLA”). The term “motif” refers to the pattern of residues in a peptideof defined length, usually about 8 to about 11 amino acids, which isrecognized by a particular MHC allele. The peptide motifs are typicallydifferent for each MHC allele and differ in the pattern of the highlyconserved residues and negative binding residues.

Neoplasia, malignancy, cancer or tumor: A neoplasm is an abnormal growthof tissue or cells that results from excessive cell division. Neoplasticgrowth can produce a tumor. The amount of a tumor in an individual isthe “tumor burden” which can be measured as the number, volume, orweight of the tumor. A tumor that does not metastasize is referred to as“benign.” A tumor that invades the surrounding tissue and/or canmetastasize is referred to as “malignant.” Examples of hematologicaltumors include leukemias, including acute leukemias (such as acutelymphocytic leukemia, acute myelocytic leukemia, acute myelogenousleukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic anderythroleukemia), chronic leukemias (such as chronic myelocytic(granulocytic) leukemia, chronic myelogenous leukemia, and chroniclymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's lymphoma (indolent and high grade forms), multiplemyeloma, Waldenstrom's macroglobulinemia, heavy chain disease,myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, includefibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy,pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostatecancer, hepatocellular carcinoma, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroidcarcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervicalcancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors(such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma).

In several examples, the malignancy is a B-cell malignancy, such as, butnot limited to non-Hodgkin's lymphoma, hairy cell leukemia or chroniclymphocytic leukemia.

Non-Hodgkin's lymphoma (NHL): Refers to a heterogeneous group of cancersprincipally arising from B lymphocytes. The subtypes of NHL aretypically grouped into three distinct categories based on theiraggressiveness, or histologic grade. These categories are indolent(low-grade), aggressive (intermediate-grade) and highly aggressive(high-grade). NHLs include, but are not limited to, diffuse large B-celllymphoma, mantle cell lymphoma, MALT lymphoma, follicular lymphoma,small lymphocytic lymphoma and Burkitt's lymphoma.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

Oligonucleotide: A linear polynucleotide sequence of up to about 100nucleotide bases in length.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition, 1975,describes compositions and formulations suitable for pharmaceuticaldelivery of the antibodies disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Polynucleotide: The term polynucleotide or nucleic acid sequence refersto a polymeric form of nucleotide at least 10 bases in length. Arecombinant polynucleotide includes a polynucleotide that is notimmediately contiguous with both of the coding sequences with which itis immediately contiguous (one on the 5′ end and one on the 3′ end) inthe naturally occurring genome of the organism from which it is derived.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector; into an autonomously replicating plasmid orvirus; or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (such as a cDNA) independent of othersequences. The nucleotides can be ribonucleotides, deoxyribonucleotides,or modified forms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (such as glycosylation orphosphorylation). In one embodiment, the polypeptide is CD22polypeptide. A “residue” refers to an amino acid or amino acid mimeticincorporated in a polypeptide by an amide bond or amide bond mimetic. Apolypeptide has an amino terminal (N-terminal) end and a carboxyterminal (C-terminal) end.

Preventing, treating or ameliorating a disease: “Preventing” a diseaserefers to inhibiting the full development of a disease. “Treating”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop,such as a B-cell malignancy. “Ameliorating” refers to the reduction inthe number or severity of signs or symptoms of a disease, such as aB-cell malignancy.

Promoter: A promoter is an array of nucleic acid control sequences thatdirects transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, forexample, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elementswhich can be located as much as several thousand base pairs from thestart site of transcription. Both constitutive and inducible promotersare included (see for example, Bitter et al., Methods in Enzymology153:516-544, 1987).

Specific, non-limiting examples of promoters include promoters derivedfrom the genome of mammalian cells (such as the metallothioneinpromoter) or from mammalian viruses (such as the retrovirus longterminal repeat; the adenovirus late promoter; the vaccinia virus 7.5Kpromoter) may be used. Promoters produced by recombinant DNA orsynthetic techniques may also be used. A polynucleotide can be insertedinto an expression vector that contains a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequenceof the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific nucleic acid sequences thatallow phenotypic selection of the transformed cells.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell. Inone embodiment, a preparation is purified such that the protein orpeptide represents at least 50%, such as at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 98%, of the totalpeptide or protein content of the preparation.

The CD22 polypeptides disclosed herein, or antibodies that specificallybind CD22, can be purified by any of the means known in the art. See forexample Guide to Protein Purification, ed. Deutscher, Meth. Enzymol.185, Academic Press, San Diego, 1990; and Scopes, Protein Purification:Principles and Practice, Springer Verlag, New York, 1982. Substantialpurification denotes purification from other proteins or cellularcomponents. A substantially purified protein is at least 60%, 70%, 80%,90%, 95% or 98% pure. Thus, in one specific, non-limiting example, asubstantially purified protein is 90% free of other proteins or cellularcomponents.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques.

Recombinant toxins: Chimeric proteins in which a cell targeting moietyis fused to a toxin (Pastan et al., Science, 254:1173-1177, 1991). Ifthe cell targeting moiety is the Fv portion of an antibody, the moleculeis termed a recombinant immunotoxin (Chaudhary et al., Nature,339:394-397, 1989). The toxin moiety is genetically altered so that itcannot bind to the toxin receptor present on most normal cells.Recombinant immunotoxins selectively kill cells which are recognized bythe antigen binding domain. These recombinant toxins and immunotoxinscan be used to treat cancer, for example, cancers in which CD22 isexpressed.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a CD22 polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 90%,95%, 96%, 97%, 98% or 99% sequence identity counted over the full lengthalignment with the amino acid sequence of the antibody using the NCBIBlast 2.0, gapped blastp set to default parameters. For comparisons ofamino acid sequences of greater than about 30 amino acids, the Blast 2sequences function is employed using the default BLOSUM62 matrix set todefault parameters, (gap existence cost of 11, and a per residue gapcost of 1). When aligning short peptides (fewer than around 30 aminoacids), the alignment should be performed using the Blast 2 sequencesfunction, employing the PAM30 matrix set to default parameters (open gap9, extension gap 1 penalties). Proteins with even greater similarity tothe reference sequences will show increasing percentage identities whenassessed by this method, such as at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% sequence identity. Whenless than the entire sequence is being compared for sequence identity,homologs and variants will typically possess at least 80% sequenceidentity over short windows of 10-20 amino acids, and may possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are available at the NCBIwebsite on the internet. One of skill in the art will appreciate thatthese sequence identity ranges are provided for guidance only; it isentirely possible that strongly significant homologs could be obtainedthat fall outside of the ranges provided.

Soluble CD22 (sCD22): A non-membrane-bound form of CD22, a 135 kDaphosphoglycoprotein adhesion molecule present on the surface of B cells,including human B cell malignancies. Soluble CD22 can be any portion ofthe CD22 protein not connected to the membrane, and is usually atruncated form of CD22. For example, sCD22 can be about 100 kDa, howeverit can also be no more than or no less than about 90, 80, 70, 60, 50,40, or 30 kDa, or smaller. Standard software is available fordetermining the transmembrane domain of CD22.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a CD22 specific binding agent is an agent thatbinds substantially to a CD22 polypeptide. In one embodiment, thespecific binding agent is a human monoclonal antibody that specificallybinds the CD22 polypeptide.

The term “specifically binds” refers, with respect to an antigen such asCD22, to the preferential association of an antibody or other ligand, inwhole or part, with a cell or tissue bearing that antigen and not tocells or tissues lacking that antigen. It is recognized that a certaindegree of non-specific interaction may occur between a molecule and anon-target cell or tissue. Nevertheless, specific binding may bedistinguished as mediated through specific recognition of the antigen.Although selectively reactive antibodies bind antigen, they may do sowith low affinity. On the other hand, specific binding results in a muchstronger association between the antibody (or other ligand) and cellsbearing the antigen than between the bound antibody (or other ligand)and cells lacking the antigen. Specific binding typically results ingreater than 2-fold, such as greater than 5-fold, greater than 10-fold,or greater than 100-fold increase in amount of bound antibody or otherligand (per unit time) to a cell or tissue bearing the CD22 polypeptideas compared to a cell or tissue lacking the polypeptide. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats are appropriate for selecting antibodies or otherligands specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow & Lane (Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York, 1988) for a description of immunoassay formatsand conditions that can be used to determine specific immunoreactivity.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and veterinary subjects, including human andnon-human mammals.

Therapeutically effective amount: A quantity of a specific substancesufficient to achieve a desired effect in a subject being treated. Forinstance, this can be the amount necessary to inhibit or suppress growthof a tumor. In one embodiment, a therapeutically effective amount is theamount necessary to eliminate a tumor. When administered to a subject, adosage will generally be used that will achieve target tissueconcentrations (for example, in tumors) that has been shown to achieve adesired in vitro effect.

Toxin: A molecule that is cytotoxic for a cell. Toxins include abrin,ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinumtoxin, saporin, restrictocin or gelonin, or modified toxins thereof. Forexample, PE and DT are highly toxic compounds that typically bring aboutdeath through liver toxicity. PE and DT, however, can be modified into aform for use as an immunotoxin by removing the native targetingcomponent of the toxin (such as domain Ia of PE or the B chain of DT)and replacing it with a different targeting moiety, such as an antibody.

Transduced: A transduced cell is a cell into which has been introduced anucleic acid molecule by molecular biology techniques. As used herein,the term transduction encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Human Monoclonal Antibodies that Specifically Bind CD22

Described herein are isolated human monoclonal antibodies specific forhuman CD22. The human monoclonal antibodies bind CD22 with adissociation constant (K_(d)) of about 25 nM or less. In someembodiments, the human monoclonal antibodies bind human CD22 with abinding affinity of about 25 nM or less, 24 nM or less, 23 nM or less,22 nM or less, 21 nM or less, 20 nM or less, about 19 nM or less, about18 nM or less, about 17 nM or less, about 16 nM or less, or about 15 nMor less. In other embodiments, the human monoclonal antibodies bindhuman CD22 with a binding affinity of about 10 nM or less, about 9 nM orless, about 8 nM or less, about 7 nM or less, about 6 nM or less, orabout 5 nM or less. In other embodiments, the human monoclonalantibodies bind human CD22 with a binding affinity of about 4 nM orless, about 3 nM or less, about 2.5 nM or less, about 2 nM or less,about 1.5 nM or less, or about 1 nM or less. In other embodiments, thehuman monoclonal antibodies bind human CD22 with a binding affinity ofabout 1 nM or less, about 0.75 nM or less, about 0.5 nM or less, about0.25 nM or less, or about 0.1 nM or less. In one example, the bindingaffinity is about 20 nM. In another example, the binding affinity isabout 2 nM.

Also described are compositions comprising the provided human monoclonalantibodies and a pharmaceutically acceptable carrier. Nucleic acidsencoding these antibodies, expression vectors comprising these nucleicacids, and isolated host cells that express the nucleic acids are alsoprovided.

Also described herein are immunoconjugates comprising the humanmonoclonal antibodies specific for human CD22. The immunoconjugates cancomprise any therapeutic agent, toxin or other moiety. In one example,the toxin is PE or a variant or fragment thereof. Compositionscomprising the immunoconjugates are also described.

Compositions comprising the human monoclonal antibodies specific forCD22 can be used for research, diagnostic and therapeutic purposes. Forexample, the human monoclonal antibodies can be used to treat a subjectdiagnosed with a B-cell malignancy, such as, but not limited to,non-Hodgkin's lymphoma, Burkitt's lymphoma, small lymphocytic lymphoma,primary effusion lymphoma, diffuse large B-cell lymphoma, splenicmarginal zone lymphoma, MALT (mucosa-associated lymphoid tissue)lymphoma, hairy cell leukemia, chronic lymphocytic leukemia and B-cellprolymphocytic leukemia. Immunoconjugates comprising the CD22 antibodiesalso can be used to treat a patient diagnosed with a B-cell malignancy.The human monoclonal antibodies can also be used to diagnose a B-cellmalignancy in a subject. For example, the human monoclonal antibodiescan be contacted with a blood sample from the patient to detect elevatedlevels of soluble CD22, or an elevated number of cells expressing CD22.The antibodies and compositions provided herein can also be used todetect a B-cell malignancy in a subject or to confirm the diagnosis of aB-cell malignancy in a patient. The antibodies can also be used to studythe biology of CD22-expressing cancers.

Disclosed herein are fully human monoclonal antibodies that specificallybind human CD22. A major limitation in the clinical use of mousemonoclonal antibodies is the development of a human anti-murine antibody(HAMA) response in the patients receiving the treatments. The HAMAresponse can involve allergic reactions and an increased rate ofclearance of the administered antibody from the serum. Various types ofmodified monoclonal antibodies have been developed to minimize the HAMAresponse while trying to maintain the antigen binding affinity of theparent monoclonal antibody. One type of modified monoclonal antibody isa human-mouse chimera in which a murine antigen-binding variable regionis coupled to a human constant domain (Morrison and Schlom, ImportantAdvances in Oncology, Rosenberg, S. A. (Ed.), 1989). A second type ofmodified monoclonal antibody is the complementarity determining region(CDR)-grafted, or humanized, monoclonal antibody (Winter and Harris,Immunol. Today 14:243-246, 1993). However, the antibodies disclosedherein are fully human; both the framework region and the CDRs are fromhuman antibodies. Thus, a HAMA is not induced when these antibodies areadministered to a human subject.

In one embodiment, the antibodies bind CD22 with a dissociation constant(K_(d)) of about 25 nM or less. In several embodiments, the humanmonoclonal antibodies bind human CD22 with a binding affinity of about25 nM, about 24 nM, about 23 nM, about 22 nM, about 21 nM about 20 nM,about 15 nM, about 10 nM, about 5 nM, about 4 nM, about 3 nM, about 2.5nM, about 2 nM, about 1.5 nM, about 1 nM or about 0.5 nM or less.

In some embodiments, the human monoclonal antibody comprises at least aportion of the heavy chain amino acid sequence set forth as SEQ ID NO: 1or SEQ ID NO: 2. In some embodiments, the human monoclonal antibodycomprises at least a portion of the light chain amino acid sequence setforth as SEQ ID NO: 2 or SEQ ID NO: 4. In some examples, the portion ofthe light chain or heavy chain comprises one or more CDRs. In particularexamples, the heavy chain of the antibody comprises amino acids 26-33 ofSEQ ID NO: 1, amino acids 51-59 of SEQ ID NO: 1 or amino acids 97-106 ofSEQ ID NO: 1, or a combination thereof; or the light chain of theantibody comprises amino acids 27-32 of SEQ ID NO: 2, amino acids 50-52of SEQ ID NO: 2 or amino acids 89-98 of SEQ ID NO: 2, or a combinationthereof; or both. In other examples, the heavy chain of the antibodycomprises amino acids 26-35 of SEQ ID NO: 3, amino acids 53-61 of SEQ IDNO: 3 or amino acids 100-113 of SEQ ID NO: 3, or a combination thereof;or the light chain of the antibody comprises amino acids 27-32 of SEQ IDNO: 4, amino acids 50-52 of SEQ ID NO: 4 or amino acids 89-97 of SEQ IDNO: 4, or a combination thereof; or both.

The monoclonal antibody can be of any isotype. The monoclonal antibodycan be, for example, an IgM or an IgG antibody, such as IgG₁ or an IgG₂.The class of an antibody that specifically binds CD22 can be switchedwith another. In one aspect, a nucleic acid molecule encoding V_(L) orV_(H) is isolated using methods well-known in the art, such that it doesnot include any nucleic acid sequences encoding the constant region ofthe light or heavy chain, respectively. The nucleic acid moleculeencoding V_(L) or V_(H) is then operatively linked to a nucleic acidsequence encoding a C_(L) or C_(H) from a different class ofimmunoglobulin molecule. This can be achieved using a vector or nucleicacid molecule that comprises a C_(L) or C_(H) chain, as known in theart. For example, an antibody that specifically binds CD22 that wasoriginally IgM may be class switched to an IgG. Class switching can beused to convert one IgG subclass to another, such as from IgG₁ to IgG₂.

Fully human monoclonal antibodies include a human framework region. Thishuman framework region can include the framework regions disclosed inone or more of SEQ ID NOs: 1-4 (these sequences include CDR sequences aswell as framework sequences). However, the framework regions can be fromanother source. Additional examples of framework sequences that can beused include the amino acid framework sequences of the heavy and lightchains disclosed in PCT Publication No. WO 2006/074071 (see, forexample, SEQ ID NOs: 1-16 of WO 2006/074071).

Antibody fragments are encompassed by the present disclosure, such asFab, F(ab′)2, and Fv which include a heavy chain and light chainvariable region and are capable of binding the epitopic determinant onCD22. These antibody fragments retain the ability to selectively bindwith the antigen. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)2 is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of anscFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). In several examples, the variable regionincluded in the antibody is the variable region of m971 or m972.

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV₂), are also contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Additional recombinant anti-CD22 human antibodies can be isolated byscreening of a recombinant combinatorial antibody library, such as a Fabphage display library (see, for example, U.S. Patent ApplicationPublication No. 2005/0123900). In some cases the phage display librariesare prepared using cDNAs of the variable regions of heavy and lightchains prepared from mRNA derived from human lymphocytes. Methodologiesfor preparing and screening such libraries are known in the art. Thereare commercially available kits for generating phage display libraries(for example, the Pharmacia Recombinant Phage Antibody System, catalogno. 27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalogno. 240612). There are also other methods and reagents that can be usedin generating and screening antibody display libraries (see, forexample, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619; PCTPublication No. WO 91/17271; PCT Publication No. WO 92/20791; PCTPublication No. WO 92/15679; PCT Publication No. WO 93/01288; PCTPublication No. WO 92/01047; PCT Publication No. WO 92/09690; Fuchs etal., Bio/Technology 9:1370-1372, 1991; Hay et al., Hum. Antibod.Hybridomas 3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989;McCafferty et al., Nature 348:552-554, 1990; Griffiths et al., EMBO J.12:725-734, 1993)

In one embodiment, to isolate additional human antibodies thatspecifically bind CD22, a human antibody that specifically binds CD22,as described herein, is first used to select human heavy and light chainsequences having similar binding activity toward CD22, such as using theepitope imprinting methods disclosed in PCT Publication No. WO 93/06213.The antibody libraries used in this method are scFv libraries preparedand screened, using methods such as those as described in PCTPublication No. WO 92/01047, McCafferty et al., Nature 348:552-554,1990; and/or Griffiths et al., EMBO J. 12:725-734, 1993 using human CD22as the antigen.

Once initial human variable light chain (VL) and variable heavy chain(VH) segments are selected, “mix and match” experiments, in whichdifferent pairs of the initially selected VL and VH segments arescreened for CD22 binding, are performed to select VL/VH paircombinations of interest. Additionally, to increase binding affinity ofthe antibody, the VL and VH segments can be randomly mutated, such aswithin H-CDR3 region or the L-CDR3 region, in a process analogous to thein vivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying VH and VL regions using PCRprimers complimentary to the H-CDR3 or L-CDR3, respectively. In thisprocess, the primers have been “spiked” with a random mixture of thefour nucleotide bases at certain positions such that the resultant PCRproducts encode VH and VL segments into which random mutations have beenintroduced into the VH and/or VL CDR3 regions. These randomly mutated VHand VL segments can be tested to determine the binding affinity forCD22.

Following screening and isolation of an antibody that binds CD22 from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (forexample, from the phage genome) and subcloned into other expressionvectors by standard recombinant DNA techniques, as described herein. Ifdesired, the nucleic acid can be further manipulated to create otherantibody fragments, also as described herein. To express a recombinanthuman antibody isolated by screening of a combinatorial library, the DNAencoding the antibody is cloned into a recombinant expression vector andintroduced into a mammalian host cell, as described herein.

IV. Immunoconjugates and Other Therapeutic Moieties

The human monoclonal antibodies specific for human CD22 described hereincan be conjugated to a therapeutic agent. Immunoconjugates include, butare not limited to, molecules in which there is a covalent linkage of atherapeutic agent to an antibody. A therapeutic agent is an agent with aparticular biological activity directed against a particular targetmolecule or a cell bearing a target molecule. One of skill in the artwill appreciate that therapeutic agents can include various drugs suchas vinblastine, daunomycin and the like, cytotoxins such as native ormodified Pseudomonas exotoxin or Diphtheria toxin, encapsulating agents(such as liposomes) which, themselves contain pharmacologicalcompositions, radioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S andother labels, target moieties and ligands.

The choice of a particular therapeutic agent depends on the particulartarget molecule or cell, and the desired biological effect. Thus, forexample, the therapeutic agent can be a cytotoxin that is used to bringabout the death of a particular target cell. Conversely, where it isdesired to invoke a non-lethal biological response, the therapeuticagent can be conjugated to a non-lethal pharmacological agent or aliposome containing a non-lethal pharmacological agent.

With the therapeutic agents and antibodies described herein, one ofskill can readily construct a variety of clones containing functionallyequivalent nucleic acids, such as nucleic acids which differ in sequencebut which encode the same EM or antibody sequence. Thus, the presentinvention provides nucleic acids encoding antibodies and conjugates andfusion proteins thereof.

Effector molecules can be linked to an antibody of interest using anynumber of means known to those of skill in the art. Both covalent andnoncovalent attachment means may be used. The procedure for attaching aneffector molecule to an antibody varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (—NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on an antibody to result in the binding of theeffector molecule. Alternatively, the antibody is derivatized to exposeor attach additional reactive functional groups. The derivatization mayinvolve attachment of any of a number of linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, Ill. The linker can beany molecule used to join the antibody to the effector molecule. Thelinker is capable of forming covalent bonds to both the antibody and tothe effector molecule. Suitable linkers are well known to those of skillin the art and include, but are not limited to, straight orbranched-chain carbon linkers, heterocyclic carbon linkers, or peptidelinkers. Where the antibody and the effector molecule are polypeptides,the linkers may be joined to the constituent amino acids through theirside groups (such as through a disulfide linkage to cysteine) or to thealpha carbon amino and carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for attaching a given agent to anantibody or other polypeptide.

The antibodies or antibody fragments disclosed herein can be derivatizedor linked to another molecule (such as another peptide or protein). Ingeneral, the antibodies or portion thereof is derivatized such that thebinding to CD22 is not affected adversely by the derivatization orlabeling. For example, the antibody can be functionally linked (bychemical coupling, genetic fusion, noncovalent association or otherwise)to one or more other molecular entities, such as another antibody (forexample, a bispecific antibody or a diabody), a detection agent, apharmaceutical agent, and/or a protein or peptide that can mediateassociate of the antibody or antibody portion with another molecule(such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by cross linking two ormore antibodies (of the same type or of different types, such as tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (such asm-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (suchas disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

A human antibody that specifically binds CD22 can be labeled with adetectable moiety. For example, the antibodies can be covalently ornon-covalently linked to a detectable label. Detectable labels suitablefor such use include any composition detectable by spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means.

Useful detection agents include fluorescent compounds, includingfluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). An antibody can also be labeled with enzymes that are useful fordetection, such as horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, glucose oxidase and the like. When an antibody islabeled with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody may also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Itshould be noted that the avidin itself can be labeled with an enzyme ora fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel may be used to detect CD22 by x-ray, emissionspectra, or other diagnostic techniques. Examples of labels forpolypeptides include, but are not limited to, the followingradioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ³¹¹I.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, radiolabels may be detected using photographicfilm or scintillation counters, fluorescent markers may be detectedusing a photodetector to detect emitted illumination. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Toxins can be employed with the CD22-specific human monoclonalantibodies described herein to produce immunotoxins. Exemplary toxinsinclude ricin, abrin, diphtheria toxin and subunits thereof, as well asbotulinum toxins A through F. These toxins are readily available fromcommercial sources (for example, Sigma Chemical Company, St. Louis,Mo.). Contemplated toxins also include variants of the toxins describedherein (see, for example, see, U.S. Pat. Nos. 5,079,163 and 4,689,401).In one embodiment, the toxin is Pseudomonas exotoxin (PE) (U.S. Pat. No.5,602,095). As used herein “Pseudomonas exotoxin” refers to afull-length native (naturally occurring) PE or a PE that has beenmodified. Such modifications can include, but are not limited to,elimination of domain Ia, various amino acid deletions in domains Ib, IIand III, single amino acid substitutions and the addition of one or moresequences at the carboxyl terminus (for example, see Siegall et al., J.Biol. Chem. 264:14256-14261, 1989). In one embodiment, the cytotoxicfragment of PE retains at least 50%, at least 75%, at least 90%, or atleast 95% of the cytotoxicity of native PE. In some examples, thecytotoxic fragment is more toxic than native PE.

Native Pseudomonas exotoxin A (PE) is an extremely active monomericprotein (molecular weight 66 kD), secreted by Pseudomonas aeruginosa,which inhibits protein synthesis in eukaryotic cells. The method of PEaction is inactivation of the ADP-ribosylation of elongation factor 2(EF-2). The exotoxin contains three structural domains that act inconcert to cause cytotoxicity. Domain 1a mediates cell binding. DomainII is responsible for translocation into the cytosol and domain IIImediates ADP ribosylation of elongation factor 2. The function of domainIb is unknown. PE employed with the monoclonal antibodies describedherein can include the native sequence, cytotoxic fragments of thenative sequence, and conservatively modified variants of native PE andits cytotoxic fragments. Cytotoxic fragments of PE include those whichare cytotoxic with or without subsequent proteolytic or other processingin the target cell. Cytotoxic fragments of PE include PE40, PE38, andPE35. For additional description of PE and variants thereof, see forexample, U.S. Pat. Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039;5,821,238; and 5,854,044; PCT Publication No. WO 99/51643; Pai et al.,Proc. Natl. Acad. Sci. USA 88:3358-3362, 1991; Kondo et al., J. Biol.Chem. 263:9470-9475, 1988; Pastan et al., Biochim. Biophys. Acta1333:C1-C6, 1997.

The CD22-specific antibodies described herein can also be used to targetany number of different diagnostic or therapeutic compounds to cellsexpressing CD22 on their surface. This can be done for research ortherapeutic purposes. Thus, an antibody of the present disclosure can beattached directly or via a linker to a drug that is to be delivereddirectly to cells expressing cell-surface CD22. Therapeutic agentsinclude such compounds as nucleic acids, proteins, peptides, amino acidsor derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, orrecombinant viruses. Nucleic acid therapeutic and diagnostic moietiesinclude antisense nucleic acids, derivatized oligonucleotides forcovalent cross-linking with single or duplex DNA, and triplex formingoligonucleotides.

Alternatively, the molecule linked to an anti-CD22 antibody can be anencapsulation system, such as a liposome or micelle that contains atherapeutic composition such as a drug, a nucleic acid (for example, anantisense nucleic acid), or another therapeutic moiety that ispreferably shielded from direct exposure to the circulatory system.Means of preparing liposomes attached to antibodies are well known tothose of skill in the art (see, for example, U.S. Pat. No. 4,957,735;Connor et al., Pharm. Ther. 28:341-365, 1985).

V. CD22 Antibody Polynucleotides and Polypeptides

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies, immunoconjugates and fusion proteins) can readily beproduced by one of skill in the art, using the amino acid sequencesprovided herein, sequences available in the art, and the genetic code.In addition, one of skill can readily construct a variety of clonescontaining functionally equivalent nucleic acids, such as nucleic acidswhich differ in sequence but which encode the same effector molecule orantibody sequence. Thus, nucleic acids encoding antibodies, conjugatesand fusion proteins are provided herein.

In some embodiments, the nucleotide sequence of the heavy chain of theCD22-specific human monoclonal antibody comprises SEQ ID NO: 7, or aportion thereof (such as a portion that encodes one or more CDRs). Inother embodiments, the nucleotide sequence of the heavy chain of theCD22-specific human monoclonal antibody comprises SEQ ID NO: 9, or aportion thereof. In some embodiments, the nucleotide sequence of thelight chain of the CD22-specific human monoclonal antibody comprises SEQID NO: 8, or a portion thereof (such as a portion that encodes one ormore CDRs). In other embodiments, the nucleotide sequence of the lightchain of the CD22-specific human monoclonal antibody comprises SEQ IDNO: 10, or a portion thereof.

Nucleic acid sequences encoding the human antibodies that specificallybind CD22 can be prepared by any suitable method including, for example,cloning of appropriate sequences or by direct chemical synthesis bymethods such as the phosphotriester method of Narang et al., Meth.Enzymol. 68:90-99, 1979; the phosphodiester method of Brown et al.,Meth. Enzymol. 68:109-151, 1979; the diethylphosphoramidite method ofBeaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phasephosphoramidite triester method described by Beaucage & Caruthers,Tetra. Letts. 22(20):1859-1862, 1981, for example, using an automatedsynthesizer as described in, for example, Needham-VanDevanter et al.,Nucl. Acids Res. 12:6159-6168, 1984; and, the solid support method ofU.S. Pat. No. 4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill wouldrecognize that while chemical synthesis of DNA is generally limited tosequences of about 100 bases, longer sequences may be obtained by theligation of shorter sequences.

Exemplary nucleic acids encoding human antibodies that specifically bindCD22 can be prepared by cloning techniques. Examples of appropriatecloning and sequencing techniques, and instructions sufficient to directpersons of skill through many cloning exercises are found in Sambrook etal., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA Chemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis,Minn.), Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories,Inc. (Palo Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life Technologies,Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (FlukaChemie AG, Buchs, Switzerland), Invitrogen (Carlsbad, Calif.), andApplied Biosystems (Foster City, Calif.), as well as many othercommercial sources known to one of skill.

Nucleic acids encoding native EM or anti-CD22 antibodies can be modifiedto form the EM, antibodies, or immunoconjugates of the presentdisclosure. Modification by site-directed mutagenesis is well known inthe art. Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In one embodiment, immunoconjugates are prepared by inserting the cDNAwhich encodes a human CD22-specific monoclonal antibody into a vectorwhich comprises the cDNA encoding the EM. The insertion is made so thatthe antibody and the EM are read in frame, that is in one continuouspolypeptide which contains a functional antibody region and a functionalEM region. In one embodiment, cDNA encoding an EM, such as a label orenzyme, is ligated to an antibody so that the EM is located at thecarboxyl terminus of the antibody. In another embodiment, the EM islocated at the amino terminus of the antibody. In another example, cDNAencoding the EM is ligated to a heavy chain variable region of anantibody, so that the EM is located at the carboxyl terminus of theheavy chain variable region. The heavy chain-variable region cansubsequently be ligated to a light chain variable region of the antibodyusing disulfide bonds. In a yet another example, cDNA encoding an EM isligated to a light chain variable region of an antibody, so that the EMis located at the carboxyl terminus of the light chain variable region.The light chain-variable region can subsequently be ligated to a heavychain variable region of the antibody using disulfide bonds.

Once the nucleic acids encoding an EM, anti-CD22 antibody, or animmunoconjugate are isolated and cloned, the desired protein can beexpressed in a recombinantly engineered cell such as bacteria, plant,yeast, insect and mammalian cells. It is expected that those of skill inthe art are knowledgeable in the numerous expression systems availablefor expression of proteins including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the Cos, CHO, HeLaand myeloma cell lines.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart. Hybridomas expressing the antibodies of interest are alsoencompassed by this disclosure.

The expression of nucleic acids encoding the isolated proteins describedherein can be achieved by operably linking the DNA or cDNA to a promoter(which is either constitutive or inducible), followed by incorporationinto an expression cassette. The cassettes can be suitable forreplication and integration in either prokaryotes or eukaryotes. Typicalexpression cassettes contain specific sequences useful for regulation ofthe expression of the DNA encoding the protein. For example, theexpression cassettes can include appropriate promoters, enhancers,transcription and translation terminators, initiation sequences, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation, and a transcription/translation terminator.For E. coli, this includes a promoter such as the T7, trp, lac, orlambda promoters, a ribosome binding site, and preferably atranscription termination signal. For eukaryotic cells, the controlsequences can include a promoter and/or an enhancer derived from, forexample, an immunoglobulin gene, SV40 or cytomegalovirus, and apolyadenylation sequence, and can further include splice donor andacceptor sequences. The cassettes can be transferred into the chosenhost cell by well-known methods such as transformation orelectroporation for E. coli and calcium phosphate treatment,electroporation or lipofection for mammalian cells. Cells transformed bythe cassettes can be selected by resistance to antibiotics conferred bygenes contained in the cassettes, such as the amp, gpt, neo and hyggenes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the Cos,CHO, HeLa and myeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein (i.e., a human CD22-specific monoclonal antibody or animmunoconjugate comprising the antibody) without diminishing itsbiological activity. Some modifications can be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, termination codons, a methionine addedat the amino terminus to provide an initiation, site, additional aminoacids placed on either terminus to create conveniently locatedrestriction sites, or additional amino acids (such as poly His) to aidin purification steps. In addition to recombinant methods, theimmunoconjugates, effector moieties, and antibodies of the presentdisclosure can also be constructed in whole or in part using standardpeptide synthesis well known in the art.

Once expressed, the recombinant immunoconjugates, antibodies, and/oreffector molecules can be purified according to standard procedures ofthe art, including ammonium sulfate precipitation, affinity columns,column chromatography, and the like (see, generally, R. Scopes, PROTEINPURIFICATION, Springer-Verlag, N.Y., 1982). The antibodies,immunoconjugates and effector molecules need not be 100% pure. Oncepurified, partially or to homogeneity as desired, if to be usedtherapeutically, the polypeptides should be substantially free ofendotoxin.

Methods for expression of antibodies and/or refolding to an appropriateactive form, including single chain antibodies, from bacteria such as E.coli have been described and are well-known and are applicable to theantibodies disclosed herein. See, Buchner et al., Anal. Biochem.205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huse et al.,Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970, andespecially as described by Buchner et al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagent N,N′-dicylohexylcarbodimide) are well known in the art.

VI. Compositions and Therapeutic Methods

Compositions are provided that include one or more of the antibodiesthat specifically bind

CD22 that are disclosed herein in a carrier. Compositions comprisingEMs, such as immunoconjugates or immunotoxins, are also provided. Thecompositions can be prepared in unit dosage forms for administration toa subject. The amount and timing of administration are at the discretionof the treating physician to achieve the desired purposes. The antibodycan be formulated for systemic or local (such as intra-tumor)administration. In one example, the antibody that specifically bindsCD22 is formulated for parenteral administration, such as intravenousadministration.

The compositions for administration can include a solution of theantibody that specifically binds CD22 dissolved in a pharmaceuticallyacceptable carrier, such as an aqueous carrier. A variety of aqueouscarriers can be used, for example, buffered saline and the like. Thesesolutions are sterile and generally free of undesirable matter. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, toxicity adjusting agents andthe like, for example, sodium acetate, sodium chloride, potassiumchloride, calcium chloride, sodium lactate and the like. Theconcentration of antibody in these formulations can vary widely, andwill be selected primarily based on fluid volumes, viscosities, bodyweight and the like in accordance with the particular mode ofadministration selected and the subject's needs.

A typical pharmaceutical composition for intravenous administrationincludes about 0.1 to 10 mg of antibody per subject per day. Dosagesfrom 0.1 up to about 100 mg per subject per day, such as 0.5 to 50 mgper day or 0.5 to 15 mg per day, may be used, particularly if the agentis administered to a secluded site and not into the circulatory or lymphsystem, such as into a body cavity or into a lumen of an organ. Actualmethods for preparing administrable compositions will be known orapparent to those skilled in the art and are described in more detail insuch publications as Remington's Pharmaceutical Science, 19th ed., MackPublishing Company, Easton, Pa. (1995).

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody solution is thenadded to an infusion bag containing 0.9% sodium chloride, USP, andtypically administered at a dosage of from 0.5 to 15 mg/kg of bodyweight. Considerable experience is available in the art in theadministration of antibody drugs, which have been marketed in the U.S.since the approval of RITUXAN® in 1997. Antibodies can be administeredby slow infusion, rather than in an intravenous push or bolus. In somecases, a higher loading dose is administered, with subsequent,maintenance doses being administered at a lower level. For example, aninitial loading dose of 4 mg/kg may be infused over a period of some 90minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kginfused over a 30 minute period if the previous dose was well tolerated.

The antibody that specifically binds CD22 can be administered to slow orinhibit the growth or replication of cells, such as malignant B cells.In these applications, a therapeutically effective amount of an antibodyis administered to a subject in an amount sufficient to inhibit theprogression of a B-cell malignancy, or to inhibit a sign or a symptom ofthe B-cell malignancy. Suitable subjects may include those diagnosedwith a B-cell malignancy, such as, but not limited to, non-Hodgkin'slymphoma, Burkitt's lymphoma, small lymphocytic lymphoma, primaryeffusion lymphoma, diffuse large B-cell lymphoma, splenic marginal zonelymphoma, MALT (mucosa-associated lymphoid tissue) lymphoma, hairy cellleukemia, chronic lymphocytic leukemia or B-cell prolymphocyticleukemia.

A therapeutically effective amount of a human CD22-specific antibodywill depend upon the severity of the disease and the general state ofthe patient's health. A therapeutically effective amount of the antibodyis that which provides either subjective relief of a symptom(s) or anobjectively identifiable improvement as noted by the clinician or otherqualified observer. These compositions can be administered inconjunction with another chemotherapeutic agent, either simultaneouslyor sequentially.

Many chemotherapeutic agents are presently known in the art. In oneembodiment, the chemotherapeutic agents is selected from the groupconsisting of mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti-survival agents,biological response modifiers, anti-hormones, e.g. anti-androgens, andanti-angiogenesis agents.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of at least one of the antibodies or immunoconjugates disclosedherein to effectively treat the patient. The dosage can be administeredonce but may be applied periodically until either a therapeutic resultis achieved or until side effects warrant discontinuation of therapy. Inone example, a dose of the antibody is infused for thirty minutes everyother day. In this example, about one to about ten doses can beadministered, such as three or six doses can be administered every otherday. In a further example, a continuous infusion is administered forabout five to about ten days. The subject can be treated at regularintervals, such as monthly, until a desired therapeutic result isachieved. Generally, the dose is sufficient to treat or amelioratesymptoms or signs of disease without producing unacceptable toxicity tothe patient.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, Pa., (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin, antibody or a drug, as a central core. In microspheresthe therapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y.,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibodycompositions disclosed herein. Various degradable and nondegradablepolymeric matrices for use in controlled drug delivery are known in theart (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, theblock copolymer, polaxamer 407, exists as a viscous yet mobile liquid atlow temperatures but forms a semisolid gel at body temperature. It hasbeen shown to be an effective vehicle for formulation and sustaineddelivery of recombinant interleukin-2 and urease (Johnston et al.,Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech.44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa. (1993)). Numerous additionalsystems for controlled delivery of therapeutic proteins are known (seeU.S. Pat. No. 5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No.4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat.No. 4,957,735; U.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S.Pat. No. 5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164;U.S. Pat. No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No.5,506,206; U.S. Pat. No. 5,271,961; U.S. Pat. No. 5,254,342 and U.S.Pat. No. 5,534,496).

VI. Diagnostic Methods and Kits

A method is provided herein for the detection of expression of CD22 invitro or in vivo. In one example, expression of CD22 is detected in abiological sample. The sample can be any sample, including, but notlimited to, tissue from biopsies, autopsies and pathology specimens.Biological samples also include sections of tissues, for example, frozensections taken for histological purposes. Biological samples furtherinclude body fluids, such as blood, serum, plasma, sputum, spinal fluidor urine. A biological sample is typically obtained from a mammal, suchas a rat, mouse, cow, dog, guinea pig, rabbit, or primate. In oneembodiment, the primate is a human.

In several embodiments, a method is provided for detecting a B-cellmalignancy such as non-Hodgkin's lymphoma, Burkitt's lymphoma, smalllymphocytic lymphoma, primary effusion lymphoma, diffuse large B-celllymphoma, splenic marginal zone lymphoma, MALT lymphoma, hairy cellleukemia, chronic lymphocytic leukemia or B-cell prolymphocyticleukemia. CD22 is expressed in 60-70% of B cell lymphomas and leukemias.Thus, CD22-specific antibodies can be used to detect the number of cellsexpressing CD22 in a blood sample from a subject to detect a B-cellmalignancy in the subject, or to confirm a diagnosis of a B-cellmalignancy in a subject.

The invention provides a method for detecting CD22 in a biologicalsample. In one embodiment, the biological sample comprises PBMCscollected from a subject. The method for detecting CD22 includescontacting a biological sample with a human antibody that binds CD22under conditions conducive to the formation of an immune complex, anddetecting the immune complex, to detect the CD22 in the biologicalsample. In one example, an increase in the number of CD22-positive cellsin the sample indicates that the subject has a B-cell malignancy. Inanother example, detecting an increase in the number of CD22-positivecells in the sample confirms a diagnosis of a B-cell malignancy in asubject.

It has also been demonstrated that soluble forms of CD22 can be detectedand quantified in body fluid samples, such as serum or blood samples,from subjects with B-cell malignancies (see, for example, U.S. PatentApplication Publication No. 2005/0244828). The major species of sCD22protein is about 100 kDa, which is smaller than the reported size of themembrane-bound CD22 antigen (135 kDa). Levels of sCD22 are elevated insubjects with B-cell malignancies, such as CLL and HCL, as compared tosCD22 levels in healthy individuals. In addition, levels of sCD22correlate well to tumor burden in subjects with CLL and HCL, and sCD22levels decrease with effective cancer therapies.

In general, relative levels of sCD22 in serum samples can be measuredagainst reference serum baselines from subjects free from B-cellmalignancy in order to provide a framework for determining normal CD22levels versus elevated CD22 levels. Thus, an elevated sCD22 level in asubject as compared to a control indicates the presence of a B-cellmalignancy.

Additionally, sCD22 levels can be monitored periodically in subjectswith B-cell malignancy as they undergo successive rounds of cancertherapy. A lowered (or lowering) sCD22 level is an indicator oftherapeutic effectiveness. Conversely, elevated sCD22 indicates that aparticular therapeutic intervention is ineffective at treating thecancer. Similarly, the absence of a decline in sCD22 is also anindication that tumor burden is not declining in response to thistherapy. Thus, sCD22 levels can be used to make decisions regardingchoice of therapy, as well as to diagnose or prognose development orprogression of a B-cell malignancy.

Soluble CD22 levels are also useful as for the screening of newanti-cancer therapeutic compounds. For instance, potential therapeuticcompounds are administered to a subject with a B-cell lymphoma orleukemia, and the therapeutic efficacy of the compounds is ascertainedby monitoring sCD22 levels in the subject over time. Efficacy isdetermined as discussed above for treatments.

In one embodiment, the human antibody that specifically binds CD22 isdirectly labeled with a detectable label. In another embodiment, thehuman antibody that specifically binds CD22 (the first antibody) isunlabeled and a second antibody or other molecule that can bind thehuman antibody that specifically binds CD22 is labeled. As is well knownto one of skill in the art, a second antibody is chosen that is able tospecifically bind the specific species and class of the first antibody.For example, if the first antibody is a human IgG, then the secondaryantibody may be an anti-human-1gG. Other molecules that can bind toantibodies include, without limitation, Protein A and Protein G, both ofwhich are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent, is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In an alternative embodiment, CD22 can be assayed in a biological sampleby a competition immunoassay utilizing CD22 standards labeled with adetectable substance and an unlabeled human antibody that specificallybinds CD22. In this assay, the biological sample, the labeled CD22standards and the human antibody that specifically bind CD22 arecombined and the amount of labeled CD22 standard bound to the unlabeledantibody is determined. The amount of CD22 in the biological sample isinversely proportional to the amount of labeled CD22 standard bound tothe antibody that specifically binds CD22.

The methods disclosed herein can be used for a number of purposes. Inone embodiment, the human antibody that specifically binds CD22 can beused to detect the production of CD22 in cells in cell culture, or theexpression of CD22 on the surface of cells in culture. In anotherembodiment, the antibody can be used to detect the amount of CD22 in abiological sample, the number of cells in a sample that express CD22, orthe level of expression of CD22 in a cell population. In one embodiment,the antibodies described herein can be used to detect the presence ofsCD22 in a biological sample, such as a blood or serum sample.

CD22 is expressed in 60-70% of all B-cell malignancies. In addition,sCD22 has been detected in the serum or blood of patients diagnosed withB-cell malignancies. In one embodiment, a kit is provided for detectingCD22 in a biological sample, such as a blood sample. For example, toconfirm a B-cell malignancy diagnosis in a subject, a blood sample canbe obtained from the subject, PBMCs from the sample isolated, and thecells evaluated for expression of CD22, particularly for the number ofcells expressing CD22. In another example, a B-cell malignancy diagnosisis confirmed by obtaining a blood sample from a subject and detectingsCD22 in the sample. An increased level of sCD22 relative to a controlsubject, or a known standard, confirms the B-cell malignancy diagnosis.

Kits for detecting a CD22 polypeptide, including cytoplasmic CD22,cell-surface CD22 and/or sCD22, will typically comprise a human antibodythat specifically binds CD22, such as any of the antibodies disclosedherein. In some embodiments, an antibody fragment, such as an Fvfragment or an Fab is included in the kit. In a further embodiment, theantibody is labeled (for example, with a fluorescent, radioactive, or anenzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds CD22. Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting CD22 in a biological sample generallyincludes the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, toa CD22 polypeptide. The antibody is allowed to specifically bind underimmunologically reactive conditions to form an immune complex, and thepresence of the immune complex (bound antibody) is detected directly orindirectly.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, magneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The antibodies can also beutilized in immunoassays such as but not limited to radioimmunoassays(RIAs), enzyme linked immunosorbent assays (ELISA), orimmunohistochemical assays. The antibodies can also be used forfluorescence activated cell sorting (FACS). A FACS employs a pluralityof color channels, low angle and obtuse light-scattering detectionchannels, and impedance channels, among other more sophisticated levelsof detection, to separate or sort cells (see U.S. Pat. No. 5,061,620).Any of the human antibodies that specifically bind CD22, as disclosedherein, can be used in these assays. Thus, the antibodies can be used ina conventional immunoassay, including, without limitation, an ELISA, anRIA, FACS, tissue immunohistochemistry, Western blot orimmunoprecipitation.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

Examples Example 1: Identification and Characterization of HumanAnti-CD22 mAbs Materials and Methods Proteins

Soluble CD22 was purchased from PeproTech (Rocky Hill, N.J.). CD22-Fcwas generated according to standard procedures. Briefly, theextracellular domain of CD22 protein was expressed as a fusion to humanIgG1 Fc using pCDNA1.1-22-Fc as described previously (Ho et al., J.Biol. Chem. 280, 607-617, 2005). The plasmid was transfected into 293Tcells by LIPOFECTAMINE™ reagent (Invitrogen, Carlsbad, Calif.), and theCD22-Fc protein was harvested from the culture supernatant and purifiedwith a protein A column (GE Healthcare, Piscataway, N.J.). Forproduction of CD22 sub-domain proteins, DNA fragments encoding variousdomains were PCR-amplified, introducing EcoRI and XhoI restriction sitesinto the 5 and 3′ ends, respectively. The DNA fragments were thendigested with both enzymes and cloned into the pSecTag2C (Invitrogen)expression vector digested with the same two enzymes. Different CD22domains were expressed by transient transfection into 293T cells usingthe POLYFECT™ transfection agent (Qiagen, Hilden, Germany) according tothe manufacturer's protocol.

Since the pSecTag vector has the murine kappa chain leader fused toN-terminus of the protein expressed from it, the resulting fusionprotein is secreted to the supernatant. In addition, all the expressedCD22 domain proteins had c-Myc and His tags from the vector fused totheir C-termini. They were used for detection in ELISA and affinitypurification. Cell culture supernatant containing the secreted CD22fragments was used directly in ELISA assay, and purified CD22 fragmentswere used for selecting antibodies targeting specific CD22 regions.Specifically, the CD22 fragment containing Ig-like domains 1 and 2 waspurified using the Ni-NTA-agarose column (Qiagen) for selectingCD22-specific antibodies targeting this region. Briefly, supernatantfrom 293T cells transfected with pSecTag2C-CD22 (domains 1+2) was bufferexchanged to PBS before it was loaded on the Ni-NTA-agarose column fornative purification according to the manufacture's protocol. The finalproduct was dialyzed against PBS. Purified CD22 fragments were used inphage panning.

Cell Lines

T lymphoma cell line SupT1 and B lymphoma cell lines Raji, BJAB, andST486 were obtained from ATCC (Manassas, Va.). SupT1 cells weremaintained in RPMI containing 10% FBS, whereas the B cell lines weremaintained in RPMI containing 20% FBS.

Selection of CD22 Antibody

Two different panning procedures were used to isolate anti-CD22 antibodyfrom a human naïve Fab library (Zhu et al., Proc. Natl. Acad. Sci. USA104:12123-12128, 2007). CD22-Fc and purified CD22 domains 1+2 werecoated directly to Maxisorp plates (Nunc, Denmark) in PBS buffer at 40°C. overnight for the plate format panning. Soluble CD22 was labeledfirst with EZ-link Sulfo-NHS-LC-Biotin (Pierce, Rockford, Ill.) for thestreptavidin-conjugated magnetic bead format panning. For the plateformat, approximately 10¹² Fabs displayed on the surface of phageamplified from the large naïve library were suspended in PBS with 2% drymilk and applied first to wells coated with irrelevant Fc fusionproteins for pre-absorption in the case of panning against CD22-Fc.After 1 hour at room temperature, phage suspension was transferred tothe wells coated with CD22-Fc. After two-hour incubation at roomtemperature, the wells were washed and the phage was rescued with TG1cells.

For panning against CD22 domains 1+2, the pre-incubation step withirrelevant Fc fusion protein was omitted. For the bead format,biotin-labeled CD22 were first incubated with the phage in 1 ml of aPBS+2% dry milk suspension at room temperature for 2 hours. Fifteen μlof DYNABEADS™ MyOne Streptavidin T1 (Invitrogen Dynal AS, Oslo, Norway)pre-blocked with PBS+2% dry milk was added to the antigen/phage mixturefor another one hour at room temperature. The beads were then washed andphage was rescued with TG1 cells. A total of four rounds were performedfor each format. Monoclonal ELISA was then performed to select forpositive clones. Two hundred clones were screened from each format. Onedominant positive clone from each procedure was finally selected.

Fab, IgG1, and scFv Expression and Purification

Plasmids of the positive clones were used to transform the E. colistrain HB2151 for Fab expression. The heavy and light chains of eachclone were transferred to the pDR12 vector for making the IgG1 format ofthe two antibodies. To make the scFv format of the antibody, the DNAfragments encoding the heavy and light chains of the clone were linkedthrough an overlapping PCR procedure. The resulting scFv gene had theheavy chain followed by a linker containing amino acids GGGGS (SEQ IDNO: 11), and the light chain. One SfiI restriction site was alsointroduced during the PCR procedure to each end of the scFv DNAfragment, which was cloned into the pComb3x vector (Scott et al.,“Phage-display vector,” In. Phage display: a laboratory manual. ColdSpring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, Barbas, C F,III, editor, 2001) digested with SfiI for protein expression. ScFvplasmid was used to transform E. coli strain HB2151 for expression. IgG1plasmids were transiently transfected to 293 free style cells(Invitrogen) for IgG1 production. Fab and scFv were purified using theNi-NTA agarose bead (Qiagen) and IgG1 with the protein A SEPHAROSE™column (GE Healthcare, Piscataway, N.J.).

ELISA

CD22 or CD22-Fc in PBS was coated to a 96-well plate overnight at 4° C.The plate was then washed with PBST (PBS+0.05% Tween) and blocked withPBST supplemented with 5% dry milk. Fab, scFv or IgG1 diluted inPBST+milk was added to the wells. The Fab and scFv binding was detectedusing the anti-His-IgG-HRP (Qiagen) and the IgG1 binding was detectedusing the anti-human Fc-IgG-HRP (Sigma, St. Louis, Mo.). After washing,ABTS substrate (Roche, Mannheim, Germany) was added to each well andOD₄₀₅ was recorded after five to ten minutes. For competition ELISA, oneantibody was first coated to the ELISA plate. CD22-Fc was mixed withindicated Fabs or scFv in the PBST+5% milk buffer and added to eachwell. The bound CD22-Fc was then detected with anti-human Fc-IgG-HRP.

Epitope Mapping

CD22 sub-domains secreted into the culture supernatant were useddirectly in an ELISA assay. Briefly, the testing antibodies and anti-Hisantibody (Qiagen) were coated to the ELISA plates. Cell culturesupernatant (100 μl) containing CD22 fragments with both His and c-Myctags were added to the pre-coated wells. After washing,anti-c-Myc-IgG-HRP was added to each well to measure the amount of CD22fragments captured either by anti-His antibody as a measurement ofexpression of each CD22 fragment, or by testing antibodies as ameasurement of their epitopes.

Flow Cytometry

SupT1, Raji, BJAB, and ST486 cells in suspension culture were collectedby centrifugation and then re-suspended in ice-cold RPMI+10% FBS mediumat a density of 1×10⁶/ml. ScFv or IgG1 at different concentrations wereadded to the cells. Cells were left on ice for half an hour before beingwashed with the same complete RPMI medium. For scFv HA22, a mixture ofmouse anti-His and anti-mouse-IgG-FITC was added to the cells foranother half an hour on ice. For IgG1, anti-human-IgG-FITC was added tothe cells. The cells were then washed with RPMI medium twice andre-suspended in PBS and subjected to flow cytometry analysis using theFACSCalibur™ (Becton Dickinson, San Jose, Calif.)

Results

Two CD22-specific Fabs were isolated. Using CD22-Fc as the panningantigen and the plate format, one dominant clone, m971, was isolated.Another clone, m972, was isolated in parallel using biotin-labeled CD22as the panning antigen and the streptavidin bead format. The heavy andlight chain amino acid sequences of m972 and m971 are shown below andare set forth herein as SEQ ID NOS: 1-4. CDR sequences are underlined.The corresponding nucleotide sequences of the heavy and light chains areset forth herein as SEQ ID NOS: 7-10.

m972 heavy chain (SEQ ID NO: 1)EVQLVQSGGGVVRPGGSLRLPCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARGGDDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAG m972 light chain (SEQ ID NO: 2)RIVMTQSPGTLSVSPGETATLSCRASQSFSNMLAWYQQKSGQPPRLLIYGVSTRAAGVPARFSGSGSGTEFTLTISNLQSEDFAVYYCQQYGDWPRYTFGQGTKVERKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECm971 heavy chain (SEQ ID NO: 3)QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVSHKPSNTKVDKKVEPKSCDKTSGQAG m971 light chain (SEQ ID NO: 4)DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC

The clones were expressed and purified in Fab, IgG1 and, in the case ofm972, scFv format (FIG. 1A). In Fab format, m972 bound CD22 ten timesbetter than m971 with apparent EC50s of 2 and 20 nM, respectively (FIG.1B), although the difference was less significant for their IgGls(EC50s<1 nM) (FIG. 1C). This was probably due to the increased valencyof IgG1 and the multimeric nature of CD22 (Han et al., Nat. Chem. Biol.1:93-97, 2005). M972 was further converted into the scFv format; it hada similar binding affinity as its Fab format and binding was comparableto that of scFv HA22 (FIG. 1D). The two Fabs both bound to CD22-Fc, butwith different profiles. As measured by ELISA, m971 bound better toCD22-Fc than both m972 and HA22, whereas m972 bound better to itsselecting antigen CD22 than m971 (FIGS. 1B, 1D and 1E). This suggeststhat soluble CD22 purified from mammalian cells and CD22-Fc expressedfrom mammalian cells may adopt different conformations. It is not clearat this point if this observation has any correlation with cell surfaceexpressed CD22, even though a report has suggested that CD22 may adopthighly flexible structures in domains 1 and 2 (Toba et al., Exp.Hematol. 30, 205-211, 2002). When a competition ELISA was performedamong the two new Fabs and HA22 scFv, no significant cross competitionwas detected among them (FIG. 1E). This suggests that the two newantibodies have distinct epitopes on CD22.

To better characterize the binding epitopes of the two antibodies,protein fragments containing different Ig-like domains of CD22 wereexpressed and evaluated in an ELISA. It was found that HA22 boundefficiently to CD22 fragments that included both domains 2 and 3, eventhough very weak binding was also detected against fragments containingonly domain 3 (FIG. 2). This suggests that the HA22 epitope residesprimarily in domain 3, and domain 2 is important in maintaining theintegrity of the epitope. This is in agreement with the published datashowing that RFB4, the parental IgG of HA22, competed partially withanother mAb that binds to domain 3 (Stein et al., Cancer Immunol.Immunother. 37, 293-298, 1993). The two new antibodies reacted only tothe region including domains 5 to 7 (FIG. 2). Since this is a ratherbroad region, it is not surprising that the two new antibodies do notcompete with each other (FIG. 1E).

To test whether the new antibodies bind to native, cell-associated CD22,a flow cytometry assay was performed with B lymphocyte cell lines Raji,BJAB and ST486 (FIG. 3). Neither IgG1 bound to the T lymphocyte supT1cells used as a negative control (FIG. 3). Both antibodies boundspecifically as IgG1 s to the B cell lines. Specifically, when used atthe same concentration (10 μg/ml), m972 bound to Raji cells better thanm971. In studies using BJAB and ST486 cells, when twice as much m971 wasadded (100 μg/ml versus 50 μg/ml of m972), similar bindings wereachieved by both (FIG. 3). These data suggest that m972 has a higheraffinity to cell surface-associated CD22 compared to m971, and CD22purified from the mammalian cells is a more accurate representation ofcell surface associated CD22 than CD22-Fc fusion proteins, at least whenthe epitopes of the IgGs are considered. It is noteworthy that theextent to which the two human IgGs bound to the T and B cell linesclosely resembled that of HA22. This is another strong indication ofspecific binding to cell surface CD22 by these antibodies.

In studies done in parallel to those for m971 and m972, another antibodythat bound purified CD22 domains 1+2 was isolated. This molecule alsorecognized CD22-Fc in both Fab and IgG format specifically, albeitweakly. This antibody was referred to as “weak CD22 binder.” Since itfailed to show specific binding to CD22 expressing B cells (FIG. 3),further investigation was terminated. However, the binding pattern ofthe weak binder suggests that the domains 1+2 region is highly flexible,and significant differences exist between the in vitro-expressed andcell surface-associated CD22 domains 1+2.

The binding characteristics of the two IgGs to cell surface-associatedCD22 was further analyzed by using increasing concentrations of bothIgGs against BJAB cells in a flow cytometry assay. It is evident thatthe binding of both IgGs to BJAB cells was saturable (FIG. 4A). This isanother indication of specific binding. IgG1 m972 had an apparentaffinity of approximately 15 nM, whereas m971 had an affinity ofapproximately 75 nM (FIG. 4B). This is in agreement with the flowcytometry data obtained with the other B cell lines showing betterbinding by m972. However, the affinities obtained through flow cytometrywere lower than those obtained through ELISA. This suggests thatdifferences exist between cell surface-associated CD22 and soluble CD22,despite the fact that soluble CD22 is a better representation thanCD22-Fc of the cell surface associated CD22.

Finally, to assess the new antibodies' ability to down modulatecell-surface CD22, a flow cytometry assay with HA22 was performed onRaji cells with or without pretreatment with the new IgGls. Compared tothe efficient internalization and down modulation of cell surface CD22by HA22 (Du et al., Cancer Res. 68:6300-6305, 2008), only slightdown-modulation was detected by IgG1m972. There was a 15%down-modulation of the cell surface CD22 after a one hour incubationwith 10 μg/ml of m972, whereas IgG1 m971 and an isotype control IgG1 hadno effect under the same experimental conditions.

Example 2: CD22-Specific Monoclonal Antibodies for Detecting a B-CellMalignancy in a Subject or Confirming the Diagnosis of a B-CellMalignancy in a Subject

This example describes the use of CD22-specific human monoclonalantibodies for the detection of a B-cell malignancy in a subject. Thisexample further describes the use of these antibodies to confirm thediagnosis of a B-cell malignancy in a subject.

Levels of soluble CD22 are elevated in subjects with B-cellmalignancies, relative to healthy subjects. Thus, detection andquantitation of sCD22 in patients diagnosed with, or suspected of havinga B-cell malignancy, can be used to detect a B-cell malignancy orconfirm the diagnosis of a B-cell malignancy in a subject. A bloodsample is obtained from the patient diagnosed with, or suspected ofhaving a B-cell malignancy. A blood sample taken from a patient thatdoes not have a B-cell malignancy is used as a control.

In this example, a sandwich ELISA is performed to detect sCD22 in theblood samples. Human monoclonal anti-CD22 antibody is immobilized on thesurface of a 96 well flat-bottomed plate by coating the plate with theantibody and incubating for 2 hours at room temperature. After washingthe plate twice with 0.02% Tween PBS (T-PBS), the plate was blocked with1% bovine serum albumin (BSA)-PBS to preclude nonspecific binding, thenwashed twice with T-PBS. The patient and control samples are added tothe wells and incubated for approximately 15-20 hours. After washingwith T-PBS three times, a second anti-CD22 antibody directly labeledwith horseradish peroxidase (HRP) is added to the plate. After threemore washes with T-PBS, 100 μl of 10,000-fold diluted Avidine-HRPsolution (Biosource) is added and incubated 1 hour at room temperature.After three more washes with T-PBS, 100 μl of TMB solution (Pierce) and100 μl of H₂O₂ are added and incubated for 5 minutes, followed by theaddition of 100 μl of 2N H₂SO₄ to stop the color development. The levelsof sCD22 are determined by measuring the OD value at 450 nm.

An increase in the level of sCD22 in the patient sample, relative to thecontrol sample, indicates that the subject has a B-cell malignancy.Thus, detection of sCD22 can be used to detect a B-cell malignancy in asubject, or confirm the diagnosis of a B-cell malignancy in a subject.

Example 3: CD22-Specific Monoclonal Antibodies for the Treatment ofB-Cell Malignancies

This example describes the use of CD22-specific human monoclonalantibodies for the treatment of B-cell malignancies that express CD22(such as, for example, non-Hodgkin's lymphoma, Burkitt's lymphoma, smalllymphocytic lymphoma, primary effusion lymphoma, diffuse large B-celllymphoma, splenic marginal zone lymphoma, MALT lymphoma, hairy cellleukemia, chronic lymphocytic leukemia and B-cell prolymphocyticleukemia). Patients diagnosed with a B-cell malignancy can be treatedaccording to standard procedures in the art (see, for example, U.S.Patent Application Publication No. 2006/0057136; DiJoseph et al., Clin.Cancer Res. 12(1):242-249, 2006; Kreitman et al., N. Engl. J. Med.345(4):241-247, 2001; and Leonard et al., Clin. Cancer Res.10:5327-5334, 2004).

In this example, patients diagnosed with a B-cell malignancy areadministered an immunoconjugate comprising a CD22-specific humanmonoclonal antibody linked to Pseudomonas exotoxin (PE). Preparation ofPE immunoconjugates has been described (see, for example, U.S. Pat. No.7,081,518 and U.S. Patent Application Publication Nos. 2005/0214304,2005/0118182 and 2007/0189962). The CD22 immunoconjugate is administeredby intravenous infusion in four doses, one dose per week. The dose ofimmunoconjugate administered to a patient varies depending on the weightand gender of the patient, and mode and time course of administration.Following treatment, patients are evaluated for cancer progression andother clinical signs of illness.

This disclosure provides fully human monoclonal antibodies specific forCD22. The disclosure further provides methods of treating or detectingcancers associated with expression of human CD22. It will be apparentthat the precise details of the methods described may be varied ormodified without departing from the spirit of the described disclosure.We claim all such modifications and variations that fall within thescope and spirit of the claims below.

1. An isolated human monoclonal antibody that specifically binds humanCD22 with a binding affinity of about 20 nM or less, wherein the heavychain of the antibody comprises amino acid residues 26-35, 53-61 and100-113 of SEQ ID NO: 3, and the light chain of the antibody comprisesamino acid residues 27-32, 50-52 and 89-97 of SEQ ID NO:
 4. 2. Theisolated human monoclonal antibody of claim 1, wherein the heavy chainof the antibody comprises SEQ ID NO: 3, or the light chain of theantibody comprises SEQ ID NO: 4, or both.
 3. The isolated humanmonoclonal antibody of claim 1, wherein the antibody is an Fab, scFv orIgG antibody.
 4. A composition comprising the human monoclonal antibodyof claim 1 in a pharmaceutically acceptable carrier.
 5. An isolatedimmunoconjugate comprising the human monoclonal antibody of claim 1 andan effector molecule.
 6. The isolated immunoconjugate of claim 5,wherein the effector molecule is a toxin or a detectable label.
 7. Theisolated immunoconjugate of claim 6, wherein the toxin comprises aPseudomonas exotoxin.
 8. The isolated immunoconjugate of claim 6,wherein the detectable label comprises a fluorescent, enzymatic, orradioactive label.
 9. A composition comprising the isolatedimmunoconjugate of claim 5 in a pharmaceutically acceptable carrier. 10.A method of treating a subject with a CD22-expressing B-cell malignancy,comprising administering to the subject a therapeutically effectiveamount of the human monoclonal antibody of claim 1, wherein the antibodyis conjugated to an agent that inhibits growth of the malignant B cellsor kills the malignant B cells, thereby treating the subject.
 11. Themethod of claim 10, wherein the agent is a toxin.
 12. The method ofclaim 11, wherein the toxin is a Pseudomonas exotoxin.
 13. A method oftreating a subject with a CD22-expressing B-cell malignancy, comprisingadministering to the subject a therapeutically effective amount of thehuman monoclonal antibody of claim 1, wherein the antibody is in theIgG1 format, thereby treating the subject.
 14. A method of determiningif a subject has a CD22-expressing B-cell malignancy, or confirming adiagnosis of a CD22-expressing B-cell malignancy in a subject,comprising: contacting a sample from the subject with the isolated humanmonoclonal antibody of claim 1; detecting binding of the antibody toCD22 in the sample; and determining that the subject has aCD22-expressing B-cell malignancy, or confirming the diagnosis of theCD22-expressing B-cell malignancy in the subject, if an increase isdetected in the binding of the antibody to CD22 in the sample ascompared to binding of the antibody to a control sample.
 15. The methodof claim 14, wherein the isolated human monoclonal antibody is directlylabeled.
 16. The method of claim 14, wherein detecting binding of theantibody to CD22 in the sample comprises: contacting a second antibodythat specifically binds the isolated human monoclonal antibody with thesample; detecting the binding of the second antibody to immune complexescomprising the human monoclonal antibody and CD22 in the sample; anddetermining that the subject has a CD22-expressing B-cell malignancy,confirming the diagnosis of the CD22-expressing B-cell malignancy in thesubject, if an increase is detected in the binding of the secondantibody to the sample as compared to binding of the second antibody toa control sample.
 17. An isolated nucleic acid molecule encoding thehuman monoclonal antibody of claim
 1. 18. The isolated nucleic acidmolecule of claim 17, wherein the nucleotide sequence encoding the heavychain of the human monoclonal antibody comprises SEQ ID NO: 9 and thenucleotide sequence encoding the light chain of the human monoclonalantibody comprises SEQ ID NO:
 10. 19. An expression vector comprisingthe isolated nucleic acid molecule of claim
 17. 20. An isolated hostcell transformed with the nucleic acid molecule of claim 17.